21 research outputs found
Sequential induction of effector function, tissue migration and cell death during polyclonal activation of mouse regulatory T-cells
The ability of CD4+Foxp3+ regulatory T-cells (Treg) to produce interleukin (IL)-10 is important for the limitation of inflammation at environmental interfaces like colon or lung. Under steady state conditions, however, few Tregs produce IL-10 ex vivo. To investigate the origin and fate of IL-10 producing Tregs we used a superagonistic mouse anti-mouse CD28 mAb (CD28SA) for polyclonal in vivo stimulation of Tregs, which not only led to their numeric expansion but also to a dramatic increase in IL-10 production. IL-10 secreting Tregs strongly upregulated surface receptors associated with suppressive function as compared to non-producing Tregs. Furthermore, polyclonally expanding Tregs shifted their migration receptor pattern after activation from a CCR7+CCR52 lymph node-seeking to a CCR72CCR5+ inflammationseeking phenotype, explaining the preferential recruitment of IL-10 producers to sites of ongoing immune responses. Finally, we observed that IL-10 producing Tregs from CD28SA stimulated mice were more apoptosis-prone in vitro than their IL-10 negative counterparts. These findings support a model where prolonged activation of Tregs results in terminal differentiation towards an IL-10 producing effector phenotype associated with a limited lifespan, implicating built-in termination of immunosuppression
On-target restoration of a split T cell-engaging antibody for precision immunotherapy
T cell-engaging immunotherapies are changing the landscape of current cancer care. However, suitable target antigens are scarce, restricting these strategies to very few tumor types. Here, we report on a T cell-engaging antibody derivative that comes in two complementary halves and addresses antigen combinations instead of single molecules. Each half, now coined hemibody, contains an antigen-specific single-chain variable fragment (scFv) fused to either the variable light (V-L) or variable heavy (V-H) chain domain of an anti-CD3 antibody. When the two hemibodies simultaneously bind their respective antigens on a single cell, they align and reconstitute the original CD3-binding site to engage T cells. Employing preclinical models for aggressive leukemia and breast cancer, we show that by the combinatorial nature of this approach, T lymphocytes exclusively eliminate dual antigen-positive cells while sparing single positive bystanders. This allows for precision targeting of cancers not amenable to current immunotherapies
Rapid Regulatory T-Cell Response Prevents Cytokine Storm in CD28 Superagonist Treated Mice
Superagonistic CD28-specific monoclonal antibodies (CD28SA) are highly effective activators of regulatory T-cells (Treg cells) in rats, but a first-in-man trial of the human CD28SA TGN1412 resulted in an unexpected cytokine release syndrome. Using a novel mouse anti-mouse CD28SA, we re-investigate the relationship between Treg activation and systemic cytokine release. Treg activation by CD28SA was highly efficient but depended on paracrine IL-2 from CD28SA-stimulated conventional T-cells. Systemic cytokine levels were innocuous, but depletion of Treg cells prior to CD28SA stimulation led to systemic release of proinflammatory cytokines, indicating that in rodents, Treg cells effectively suppress the inflammatory response. Since the human volunteers of the TGN1412 study were not protected by this mechanism, we also tested whether corticosteroid prophylaxis would be compatible with CD28SA induced Treg activation. We show that neither the expansion nor the functional activation of Treg cells is affected by high-dose dexamethasone sufficient to control systemic cytokine release. Our findings warn that preclinical testing of activating biologicals in rodents may miss cytokine release syndromes due to the rapid and efficacious response of the rodent Treg compartment, and suggest that polyclonal Treg activation is feasible in the presence of antiphlogistic corticosteroid prophylaxis
Immuntherapie allergischer Erkrankungen in einem Mausmodell für allergische Atemwegsentzündungen
Allergische Erkrankungen sind Störungen, bei denen es zu Immunfehlregulationen kommt und die bei empfänglichen Individuen zur Entstehung von Allergen spezifischen T-Helfer 2 (TH2) Immunantworten führen. Neuere Untersuchungen deuten darauf hin, dass die für Soforttypallergien charakteristischen TH2 Immunantworten sowohl durch natürlich vorkommende CD4+CD25+ regulatorische T Zellen (Treg) als auch durch Antigen induzierte IL-10-secreting CD4+ regulatorische T Zellen kontrolliert werden können. Weiterhin gibt es Hinweise, dass eine erfolgreiche Allergen spezifische Immuntherapie über die Induktion von IL-10 sezernierenden T reg Zellen vermittelt wird. In ersten Teil der Arbeit wird die Effizienz einer Allergen spezifischen Immuntherapie (SIT) in einem Mausmodel für allergische Atemwegsentzündung demonstriert. Als Allergieparameter wurden Allergen spezifisches IgE im Serum, verschiedene TH1 und TH2 Cytokine in der brochoalveolären Lavage Flüssigkeit und nach in vitro Restimulation in Milzzellen untersucht. Weiterhin wurden Histologien von Lungengewebe angefertigt, um das eosinophile Entzündungsinfiltrat und die Asthma typische Becherzellmetaplasie darzustellen. Weiterhin wurden durch FACS Untersuchungen regulatorische T Zellen nachgewiesen. Es konnte gezeigt werden, dass im Mausmodell die intranasale Applikationsform der SIT die allergischen Symptome effizienter bekämpfen konnte, als die beim Menschen etablierte subcutane Applikationsform. Um Mechanismen zu definieren die eine SIT effizienter machen könnten wurde ein IL-4/IL13 Inhibitor (QY) als Adjuvans für die SIT benutzt. Für den Zytokininhibitor konnte gezeigt werden, dass bei einer Applikation während der allergischen Sensibilisierung die Entstehung einer TH2 Immunantwort und die Ausbildung allergischer Symptome verhindert wird. Die Applikation des Inhibitors zusammen mit einer SIT zeigte jedoch keine zusätzlichen signifikanten antiallergischen Effekte im Vergleich zur Durchführung der SIT als Monotherapie. Diese Ergebnisse deuten möglicherweise daraufhin , dass der bekannte Wechsel einer TH2 Immunantwort zu einer TH1 Antwort während der SIT nicht der Schlüsselmechanismus zu einer erfolgreichen Behandlung ist. Insbesondere weil unter der SIT auch in unserem Mausmodell die Induktion von IL-10 sezernierenden CD4+ T regulatorischen Zellen mit der Suppression der allergischen Atemwegsentzüdnung vergesellschaftet waren, so dass möglicherwiese diese Zellen für den Therapieerfolg relevant sind . Um die Rolle regulatorischer T Zellen im Allergiemodell näher zu beleuchten wurde im 2. Teil der Arbeit ein monoklonaler superagonistischer anti-CD28 Antikörper benutzt, von dem bekannt ist dass T regulatorische Zellen in vivo induziert werden. Es konnte gezeigt werden, dass die Applikation des Antikörpers während der allergischen Sensibilisierung die Etablierung einer TH2 Immunantwort verstärkte. Im Gegensatz dazu wurden durch die therapeutische Applikation des anti CD28 Antikörpers in einer etablierten Allergie, IL-10 sezernierende CD4+CD25+ T Zellen induziert, welches mit einer Abschwächung der gemessenen Allergieparameter einherging.Allergic disease are inflammatory disorders in which aberrant immune regulation occurs, and susceptible individuals mount allergen specific T helper 2 (Th2) responses, which drives disease pathology. Recent studies indicate that Th2 responses that are characteristic of allergic manifestations can be regulated by both naturally occurring CD4+CD25+ regulatory (Treg) cells and antigen-driven IL-10-secreting CD4+ regulatory T cells. Evidence is also emerging that successful Allergen specific immunotherapy (SIT) might work through the induction of IL-10-secreting regulatory T cells. In the first part of this work, I demonstrated the efficiency of allergen specific immunotherapy in the mouse model for allergic airway inflammation. Here I could show that intranasal administration of SIT abrogates allergic symptoms more efficiently, than the subcutaneous treatment. Furthermore, an IL-4/IL-13 (QY) inhibitor was used as an adjuvant for SIT, which has been demonstrated to have an anti-allergic potential, when administered prophylactically during allergic sensitization. However, the combination therapy with SIT and the inhibitory molecule QY did not show any significant enhancement in regards to all measured allergic parameters, when compared to monotherapy with SIT. These results provide the evidence, that shift from Th2 to Th1 cytokine profile might not be a key event in successful SIT. Subsequently, the investigation of immune mechanisms under successful SIT demonstrate that the increase of IL-10 secreting CD4+ T regulatory cells is associated with the suppression of airway inflammation in our mouse system, suggesting that these T cell subsets might be involved in the regulatory mechanisms of allergic disorders. In agreement with these findings is the second part of this work, where superagonistic a-CD28 mAb´s were used for the expansion of T regulatory cell subsets in our murine model for allergic airway inflammation. Here I could show, that the application of a-CD28 mAb during allergic sensitization, resulted in the establishment of a Th2 state, rather than a stimulation of a Treg cell population, supporting the Th2 promoting role of a-CD28 mAb together with TCR engagement. However, interesting findings were obtained by application of the superagonistic a-CD28 mAb in the challenge phase in established allergy. Conversely to the previous experiment, therapeutic administration of a-CD28 mAb lead to the generation of IL-10 secreting CD4+CD25+ T cell population in line with the induction of anti-allergic effects. Taking together the results of this study argue for the anti-inflammatory properties of T regulatory cells in allergic disease and highlights importance of these T cell subsets in the suppression of Th2 cell-driven response to allergen. Moreover, these observations suggest that the induction of IL-10 in vivo by T regulatory cells may represent a novel treatment strategy for allergic disorders
Panobinostat induces CD38 upregulation and augments the antimyeloma efficacy of daratumumab.
Phenotype of IL-10<sup>+</sup> and IL-10<sup>−</sup> Tregs 3 days after CD28SA treatment.
<p>ΔMFI (mean fluorescence intensity) ( = MFI(marker)−MFI(isotype)) of memory and Treg specific markers on unstimulated or on day 3 stimulated CD4<sup>+</sup> T-cell subsets defined by Foxp3 and/or IL-10 expression. Graphs show means ± SD from 4–8 mice and results are representative of two independent experiments. * p<0.05, ** p<0.005, *** p<0.001, ns: no significant difference.</p
IL-10 producing Tregs are apoptosis prone.
<p>(<b>A</b>) CD4<sup>+</sup>CD25<sup>+</sup> Tregs from CD28SA treated DEREG mice were isolated and stimulated over night with anti-CD3, anti-CD28, IL-2. Next day cells were stained for IL-10 and separated into IL-10<sup>+</sup>Foxp3<sup>+</sup> (black line) and IL-10<sup>−</sup>Foxp3<sup>+</sup> (grey line) via FACS Sorter and cultured for 1 to 3 days with anti-CD3, anti-CD28, IL-2 and examined for Annexin V and 7AAD positive cells. As control unseparated CD4<sup>+</sup>CD25<sup>+</sup> T-cells (dashed line) were used. Graphs show means ± SD from 3 individual experiments. ** p<0.005 compared IL-10<sup>+</sup>Foxp3<sup>+</sup> to IL-10<sup>−</sup>Foxp3<sup>+</sup> cells. (<b>B</b>) Flow cytometric analysis of costaining of Annexin V and clone 10-7A (phospholipids). (<b>C</b>) Binding of clone 10-7A given in ΔMFI ( = MFI(marker)−MFI(isotype)) on gated CD4<sup>+</sup> T-cells 3 days after CD28SA injection dependent on IL-10 and Foxp3 expression. Graphs show means ± SD from 3 or 5 mice assayed individually and results are representative of two independent experiments. ** p<0.005. (<b>D</b>) Gene expression data given in M (ln(fold change)) of functional cluster “apoptosis” (DAVID) of sorted IL-10<sup>+</sup> and IL-10<sup>−</sup> CD4<sup>+</sup>CD25<sup>+</sup> Tregs 3 days after stimulation. Transcripts selected for genes that showed ≥2-fold change and an adjusted p-value<0.001 (n = 2). (<b>E</b>) Bcl-2 expression of gated CD4<sup>+</sup> T-cells 3 days after CD28SA injection dependent on IL-10 and Foxp3 expression. * p<0.05. Results are representative of three independent experiments with 3–4 mice.</p
Migration potential of Tregs after CD28SA injection.
<p>(<b>A</b>) Flow cytometric analysis of homing related receptors in IL-10<sup>+</sup>Foxp3<sup>+</sup> (black line), IL-10<sup>−</sup>Foxp3<sup>+</sup> (grey line) and total Foxp3<sup>+</sup> (dashed line) Tregs of mice 0 to 4 days after CD28SA treatment. (<b>B</b>) Migration of Tregs <i>in vivo</i>. A small piece of sponge containing heat-killed <i>Listeria monocytogenes</i> was placed under the skin of C57BL/6 mice. After 4 days mice were treated with CD28SA or PBS. On day 7 cells washed out of sponges were stained for CD4, IL-10 and Foxp3 and compared with CD4<sup>+</sup> gated LN cells. Graphs show means ± SD from 3 mice assayed individually and results are representative of two independent experiments.</p
Suppressive activity of Treg subsets.
<p>(<b>A</b>) Isolated CD4<sup>+</sup>CD25<sup>+</sup> Tregs from CD28SA treated mice were analysed for IL-10 production using IL-10 Secretion Assay. FACS separated IL-10<sup>+</sup> (solid black line) and IL-10<sup>−</sup> CD4<sup>+</sup>CD25<sup>+</sup> Tregs (dashed black line) were incubated with CFSE-labeled naïve CD4<sup>+</sup>CD25<sup>−</sup> effector T-cells (T<sub>eff</sub>), (ratio T<sub>eff</sub>∶ T<sub>reg</sub> 1∶1 to 81∶1) and irradiated APC in the presence of anti-CD3 for 3 days. Stimulated total CD4<sup>+</sup>CD25<sup>+</sup> Tregs (solid grey line) and CD4<sup>+</sup>CD25<sup>+</sup> Tregs from untreated mice (dashed grey line) were used as controls. (<b>B</b>) Bar graph of IL-10<sup>+</sup> (white bar) and IL-10<sup>−</sup> CD4<sup>+</sup>CD25<sup>+</sup> Tregs (black bar) with statistical analysis. (<b>C</b> and <b>D</b>) like (<b>A</b> and <b>B</b>) but with FACS sorted Foxp3<sup>+</sup> of unstimulated and CD28SA stimulated DEREG mice. (<b>E</b>) Suppression is IL-10 independent. Blocking anti-IL-10R mAb (10 µg/ml, grey line) was added to cultures with stimulated (solid line) and unstimulated total CD4<sup>+</sup>CD25<sup>+</sup> Tregs (dashed line). Graphs show average numbers of cell division (acd, mean ± SD) from 3 wells assayed individually and results are representative of three independent experiments. * p<0.05, ** p<0.005, *** p<0.001, ns: no significant difference.</p